SB347 sperm size

To release the sperm and measure their size, ten animals of each sex were cut using a 22 gauge needle in the presence of 5-10 µL drop of sperm buffer (90 mM NaCl, 50 mM KCl, 2 mM MgCl2, 10 mM CaCl2, 100 mM HEPES, and 20 mM dextrose to pH 7.8 for a 2X solution) 34. Males were cut at the level of the vas deferens and hermaphrodites at the level of the spermatheca 34. Immediately following the dissections, the slides were examined using DIC optics on a Carl Zeiss 510 confocal microscope. To improve the adhesion of the sperm cells to the slide, slides were pre-treated with Poly-L-Lysine. A 4-5 µL drop of 100% Poly-L-Lysine was placed at the center of the charged surface of a ColorFrost Plus slide. A second slide was placed on top of it in such a way that both charged surfaces were facing each other and a thin film of liquid formed between them. Once the film was formed, slides were separated by sliding them over each other. Subsequently, slides were baked at 60 °C for 1-2 h. To identify the sperm, which were often very small, we added Hoechst 33342 (Sigma-Aldrich) to the sperm media at a final concentration of 100 μg/mL. Hoechst helped to visualize and identify the characteristic condensed shape of the sperm nucleus. Sperm size was measured as the cross sectional area of the spermatid using the Carl Zeiss confocal software.